Abcam is committed to meeting high standards of ethical manufacturing and has decided to discontinue this product by June 2019 as it has been generated by the ascites method. We are sorry for any inconvenience this may cause. We would recommend antibody ab1238 as a replacement.

Our RabMAb® technology is a patented hybridoma-based technology for making rabbit monoclonal antibodies. For details on our patents, please refer to RabMab® patents

We are constantly working hard to ensure we provide our customers with best in class antibodies. As a result of this work we are pleased to now offer this antibody in purified format. We are in the process of updating our datasheets. The purified format is designated 'PUR' on our product labels. If you have any questions regarding this update, please contact our Scientific Support team.

Choosing an antibody | Abcam

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Application

Antibody datasheets list the applications we have successfully tested the antibody in. When an antibody is tested in an application and fails, this is also noted on the datasheet. If an application is not listed, it means that we have not tested it and it is unknown how the antibody will perform.

Out antibodies are continuously tested in-house and datasheets are updated with latest application information. For assistance with your application, browse our protocol library.

​Nature of sample

The nature of your sample will determine which antibody is most appropriate. Consider the following aspects:

The region of the protein that you wish to detect

Antibodies are generated by immunizing host animals with an immunogenic substance. Immunogens can be full-length proteins, protein fragments, peptides, whole organisms (for example, bacteria), or cells. The immunogen is generally described on the datasheet (in some cases an exact description of the immunogen is not given for proprietary reasons).

Check that the immunogen is identical to or contained within the region of the protein you are trying to detect. For example, if you are trying to detect a cell surface protein on live cells by FACS, choose an antibody that is raised against an extracellular domain of the protein.

Sample processing

Some antibodies require samples to be treated in a specific manner. Many antibodies will only recognize proteins that have been reduced and denatured, because this reveals epitopes that would otherwise be obscured by secondary and tertiary folding of the proteins. On the other hand, some antibodies will only recognize epitopes on proteins in their native, folded state. Our antibodies for western blotting require the samples to be reduced and denatured unless otherwise noted on the datasheet.

For immunohistochemistry, some antibodies are only appropriate for unfixed frozen tissue. Others cannot bind to their targets in formalin-fixed, paraffin-embedded tissues without an antigen retrieval step that reverses the cross-links introduced by formalin fixation. These restrictions on use are noted in the applications section of the datasheets.

Sample species

If possible, choose an antibody that has been raised against the same species your sample is from.

The antibody may react with the same target protein from other species sharing sufficient amino acid sequence homology. If your sample is not from one of the species listed in the datasheet, this means that the species has not been tested and we cannot demonstrate suitability. A prediction of cross-reactivity is made based on sequence similarity.

​Choosing the species of primary antibody host

The species the primary antibody is raised in should be different from the species of your sample. This is to avoid cross-reactivity of the secondary anti-immunoglobulin antibody with endogenous immunoglobulins in the sample. For instance, if you are studying a mouse protein, choose a primary antibody that is raised in a species other than mouse. A primary antibody raised in rabbit would be an appropriate choice, followed by an anti-rabbit IgG secondary antibody.

For techniques using samples that do not contain endogenous immunoglobulin (IgG), the choice of host species of the primary antibody is less critical. An example is western blotting of a cell lysate that is not expected to contain IgG. However, tissue lysates and tissue culture supernatants that contain serum will contain immunoglobulins. IgG will appear in western blots of reduced, denatured samples as bands at 50 and 25 kDa corresponding to the heavy and light chains of the IgG molecule.

​Choosing a secondary antibody

Secondary antibodies should be against the host species of the primary antibody you are using. For example, if your primary is a mouse monoclonal, you will require an anti-mouse secondary. Check the datasheet of the secondary antibody to ensure it is tested in the application you will be using.

​Choosing antibodies for dual staining

Double immunostaining of cell cultures or tissue requires the primary antibodies to be raised in different species, and that the secondary antibodies recognize one of the species exclusively. Choose from our range of secondary anti-Ig antibodies which have been pre-adsorbed against immunoglobulins from other species to remove cross-reactivity. Alternatively, our directly conjugated primary antibodies remove the need for secondary antibodies.

​Fluorochrome and chromagen labels

Labels are conjugated to antibodies to visualize presence of the target protein. The choice of label depends on the experimental application.

Fluorescent labels emit light in the visual range when excited by light of a specific wavelength. There are several available, all with their own excitation and emission characteristics.

Enzymatic labels horseradish peroxidase (HRP) and alkaline phosphatase (AP) form a colored precipitate when combined with the appropriate substrate.

Biotinylated antibodies are useful for amplification of signal when followed by an avidin-biotin-enzyme or fluorochrome complex (commonly abbreviated as ABC reagent), or avidin or streptavidin conjugated to an enzyme or fluorochrome.

See advantages of Alexa Fluor® secondary antibodies

Get fluorochrome chart

​Other useful resources

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Direct vs indirect immunofluorescence | Abcam

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Understand the difference between direct and indirect methods for immunofluorescence.

​​Immunofluorescence (IF) or cell imaging techniques rely on the use of antibodies to label a specific target antigen with a fluorescent dye (also called fluorophores or fluorochromes) such as fluorescein isothiocyanate (FITC). Antibodies that are chemically conjugated to fluorophores are commonly used in IF.

​The fluorophore allows visualization of the target distribution in the sample under a fluorescent microscope (eg epifluorescence and confocal microscopes). We distinguish between two IF methods depending on whether the fluorophore is conjugated to the primary or the secondary antibody:

Direct IF uses a single antibody directed against the target of interest. The primary antibody is directly conjugated to a fluorophore.

Indirect IF uses two antibodies. The primary antibody is unconjugated and a fluorophore-conjugated secondary antibody directed against the primary antibody is used for detection.

The diagram below represents both direct and indirect methods.

​​

​​Both methods have their advantages and disadvantages as shown in the table below.

Direct

Indirect

Time

Protocols for direct IF are usually shorter as they only require one labeling step.

The fact that you have to use a conjugated secondary antibody to detect the primary antibody results in additional steps.

Cost

Conjugated primary antibodies are usually more expensive than their unconjugated counterparts.

Secondary antibodies are relatively inexpensive compared to primary antibodies. Further cost savings may be made by using the same conjugated secondary antibody to detect different primary antibodies.

Complexity

Fewer steps in the protocol simplify direct methods.

Added complexity in indirect methods may result from having to select the appropriate secondary antibody. This is particularly relevant in multiplex experiments where several secondary antibodies, each targeting a different species and conjugated to different dyes, are needed.

The possibility of using different conjugated secondary antibodies adds greater flexibility.

Sensitivity

The signal obtained in direct methods may seem weak when compared to indirect methods as signal amplification provided by the use of secondary antibodies does not occur.

Several secondary antibodies will bind to the primary antibody resulting in an amplified signal.

Species cross-reactivity

Species cross-reactivity is minimized in direct methods as the fluorophore is already conjugated to the primary antibody.

Secondary antibodies may cross-react with species other than the target. The use of pre-adsorbed secondary antibodies can prevent cross-reactivity.

Background

Non-specific binding is reduced through the use of conjugated primary antibodies.

Samples with endogenous immunoglobulins may exhibit a high background with indirect methods.

Direct and indirect methods are not limited to immunofluorescence. They are also relevant to other techniques that rely on the use of fluorophore-conjugated antibodies such as flow cytometry, ELISA, western blot and immunohistochemistry.

Detection of low abundance proteins can be sometimes challenging even with indirect methods. Biotinylated antibodies offer an extra layer for increased signal amplification. Learn more about how methods based on the use of biotin-conjugated antibodies work here.

Alexa Fluor® is a registered trademark of Life Technologies. Alexa Fluor® dye conjugates contain(s) technology licensed to Abcam by Life Technologies.

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Anti-Integrin beta 1 antibody (ab183666)

Function

Integrins alpha-1/beta-1, alpha-2/beta-1, alpha-10/beta-1 and alpha-11/beta-1 are receptors for collagen. Integrins alpha-1/beta-1 and alpha-2/beta-2 recognize the proline-hydroxylated sequence G-F-P-G-E-R in collagen. Integrins alpha-2/beta-1, alpha-3/beta-1, alpha-4/beta-1, alpha-5/beta-1, alpha-8/beta-1, alpha-10/beta-1, alpha-11/beta-1 and alpha-V/beta-1 are receptors for fibronectin. Alpha-4/beta-1 recognizes one or more domains within the alternatively spliced CS-1 and CS-5 regions of fibronectin. Integrin alpha-5/beta-1 is a receptor for fibrinogen. Integrin alpha-1/beta-1, alpha-2/beta-1, alpha-6/beta-1 and alpha-7/beta-1 are receptors for lamimin. Integrin alpha-4/beta-1 is a receptor for VCAM1. It recognizes the sequence Q-I-D-S in VCAM1. Integrin alpha-9/beta-1 is a receptor for VCAM1, cytotactin and osteopontin. It recognizes the sequence A-E-I-D-G-I-E-L in cytotactin. Integrin alpha-3/beta-1 is a receptor for epiligrin, thrombospondin and CSPG4. Alpha-3/beta-1 may mediate with LGALS3 the stimulation by CSPG4 of endothelial cells migration. Integrin alpha-V/beta-1 is a receptor for vitronectin. Beta-1 integrins recognize the sequence R-G-D in a wide array of ligands. Isoform 2 interferes with isoform 1 resulting in a dominant negative effect on cell adhesion and migration (in vitro). When associated with alpha-7/beta-1 integrin, regulates cell adhesion and laminin matrix deposition. Involved in promoting endothelial cell motility and angiogenesis. Involved in osteoblast compaction through the fibronectin fibrillogenesis cell-mediated matrix assembly process and the formation of mineralized bone nodules. May be involved in up-regulation of the activity of kinases such as PKC via binding to KRT1. Together with KRT1 and RACK1, serves as a platform for SRC activation or inactivation. Plays a mechanistic adhesive role during telophase, required for the successful completion of cytokinesis. Integrin alpha-3/beta-1 provides a docking site for FAP (seprase) at invadopodia plasma membranes in a collagen-dependent manner and hence may participate in the adhesion, formation of invadopodia and matrix degradation processes, promoting cell invasion. ITGA4:ITGB1 binds to fractalkine (CX3CL1) and may act as its coreceptor in CX3CR1-dependent fractalkine signaling (PubMed:23125415, PubMed:24789099). ITGA4:ITGB1 and ITGA5:ITGB1 bind to PLA2G2A via a site (site 2) which is distinct from the classical ligand-binding site (site 1) and this induces integrin conformational changes and enhanced ligand binding to site 1 (PubMed:18635536, PubMed:25398877). ITGA5:ITGB1 acts as a receptor for fibrillin-1 (FBN1) and mediates R-G-D-dependent cell adhesion to FBN1 (PubMed:12807887, PubMed:17158881).Isoform 5: Isoform 5 displaces isoform 1 in striated muscles.(Microbial infection) Integrin ITGA2:ITGB1 acts as a receptor for human echoviruses 1 and 8 (PubMed:8411387). Acts as a receptor for cytomegalovirus/HHV-5 (PubMed:20660204). Acts as a receptor for Epstein-Barr virus/HHV-4 (PubMed:17945327). Integrin ITGA5:ITGB1 acts as a receptor for human parvovirus B19 (PubMed:12907437). Integrin ITGA2:ITGB1 acts as a receptor for human rotavirus (PubMed:12941907). Acts as a receptor for mammalian reovirus (PubMed:16501085). In case of HIV-1 infection, integrin ITGA5:ITGB1 binding to extracellular viral Tat protein seems to enhance angiogenesis in Kaposi's sarcoma lesions (PubMed:10397733).

Cell membrane, sarcolemma. Cell junction. In cardiac muscle, isoform 5 is found in costameres and intercalated disks and Cell membrane. Cell projection, invadopodium membrane. Cell projection, ruffle membrane. Recycling endosome. Melanosome. Cleavage furrow. Cell projection, lamellipodium. Cell projection, ruffle. Cell junction, focal adhesion. Cell surface. Isoform 2 does not localize to focal adhesions. Highly enriched in stage I melanosomes. Located on plasma membrane of neuroblastoma NMB7 cells. In a lung cancer cell line, in prometaphase and metaphase, localizes diffusely at the membrane and in a few intracellular vesicles. In early telophase, detected mainly on the matrix-facing side of the cells. By mid-telophase, concentrated to the ingressing cleavage furrow, mainly to the basal side of the furrow. In late telophase, concentrated to the extending protrusions formed at the opposite ends of the spreading daughter cells, in vesicles at the base of the lamellipodia formed by the separating daughter cells. Colocalizes with ITGB1BP1 and metastatic suppressor protein NME2 at the edge or peripheral ruffles and lamellipodia during the early stages of cell spreading on fibronectin or collagen. Translocates from peripheral focal adhesions sites to fibrillar adhesions in a ITGB1BP1-dependent manner. Enriched preferentially at invadopodia, cell membrane protrusions that correspond to sites of cell invasion, in a collagen-dependent manner. Localized at plasma and ruffle membranes in a collagen-independent manner.

HRP Secondary Antibodies | Abcam

Western blot - Goat Anti-Rabbit HRP (IgG H&L) (ab97051)

HeLa (Human epithelial carcinoma cell line) whole cell lysate (10 µg) was loaded in each lane. All lanes were incubated with anti-beta Actin antibody (ab8227) at 1 µg/ml as the primary antibody. Goat Anti-Rabbit HRP (IgG H&L) (ab97051) was used as the secondary antibody at 1/2000 dilution (lanes 1and 2), 1/10000 dilution (lanes 3 and 4) and 1/20000 dilution (lanes 5 and 6). The blot was developed using the ECL technique and performed under reducing conditions. Exposure time was 10 seconds.

Our secondary antibody selection guide will help you choose the most appropriate secondary antibody for your experiment. Read it now to learn more about immunoglobulins classes and subclasses, preadsorbed secondary antibodies and F(ab) or F(ab’)2 fragment secondary antibodies.

* Preadsorbed (cross-adsorbed) antibodies go through an extra purification step to minimize species cross-reactivity and background. The process involves passaging of the antibody solution through a column containing immobilized serum proteins from potentially cross reactive species. Nonspecific cross-reactive secondary antibodies remain bound to the proteins in the column, while highly specific secondary antibodies are recovered from the flow through.

Our secondary antibody selection guide will help you choose the most appropriate secondary antibody for your experiment. Read it now to learn more about immunoglobulins classes and subclasses, preadsorbed secondary antibodies and F(ab) or F(ab’)2 fragment secondary antibodies.

* Preadsorbed (cross-adsorbed) antibodies go through an extra purification step to minimize species cross-reactivity and background. The process involves passaging of the antibody solution through a column containing immobilized serum proteins from potentially cross reactive species. Nonspecific cross-reactive secondary antibodies remain bound to the proteins in the column, while highly specific secondary antibodies are recovered from the flow through.

Our secondary antibody selection guide will help you choose the most appropriate secondary antibody for your experiment. Read it now to learn more about immunoglobulins classes and subclasses, preadsorbed secondary antibodies and F(ab) or F(ab’)2 fragment secondary antibodies.

* Preadsorbed (cross-adsorbed) antibodies go through an extra purification step to minimize species cross-reactivity and background. The process involves passaging of the antibody solution through a column containing immobilized serum proteins from potentially cross reactive species. Nonspecific cross-reactive secondary antibodies remain bound to the proteins in the column, while highly specific secondary antibodies are recovered from the flow through.

Our secondary antibody selection guide will help you choose the most appropriate secondary antibody for your experiment. Read it now to learn more about immunoglobulins classes and subclasses, preadsorbed secondary antibodies and F(ab) or F(ab’)2 fragment secondary antibodies.

* Preadsorbed (cross-adsorbed) antibodies go through an extra purification step to minimize species cross-reactivity and background. The process involves passaging of the antibody solution through a column containing immobilized serum proteins from potentially cross reactive species. Nonspecific cross-reactive secondary antibodies remain bound to the proteins in the column, while highly specific secondary antibodies are recovered from the flow through.

Our secondary antibody selection guide will help you choose the most appropriate secondary antibody for your experiment. Read it now to learn more about immunoglobulins classes and subclasses, preadsorbed secondary antibodies and F(ab) or F(ab’)2 fragment secondary antibodies.

* Preadsorbed (cross-adsorbed) antibodies go through an extra purification step to minimize species cross-reactivity and background. The process involves passaging of the antibody solution through a column containing immobilized serum proteins from potentially cross reactive species. Nonspecific cross-reactive secondary antibodies remain bound to the proteins in the column, while highly specific secondary antibodies are recovered from the flow through.

Our secondary antibody selection guide will help you choose the most appropriate secondary antibody for your experiment. Read it now to learn more about immunoglobulins classes and subclasses, preadsorbed secondary antibodies and F(ab) or F(ab’)2 fragment secondary antibodies.

* Preadsorbed (cross-adsorbed) antibodies go through an extra purification step to minimize species cross-reactivity and background. The process involves passaging of the antibody solution through a column containing immobilized serum proteins from potentially cross reactive species. Nonspecific cross-reactive secondary antibodies remain bound to the proteins in the column, while highly specific secondary antibodies are recovered from the flow through.